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IOSR Journal Of Pharmacy
(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219
www.iosrphr.org Volume 4, Issue 5 (May 2014), PP. 61-67
Cardiotonic activity of Parotoid gland secretion of common
Indian Toad Bufo melanostictus on isolated heart of Frog
Raju Neerati 1, Ankaiah Meesala2, Samatha Talari 3, Prasad Neerati 2 and
Venkaiah Yanamala 1*
1
Department of Zoology, 2Department of Pharmacology, 3Biotechonology Research Group,
Kakatiya University, Warangal-506009 (AP), India.
ABSTRACT:The present investigation has been undertaken to study the cardiotonic activity of parotoid gland
secretion of common Indian Toad Bufo melanostictus on isolated perfused frog heart (Rana tigrina).
Cardiotonic activity of parotoid gland secretion of B. melanostictus in normal as well as when induced with
paraoxon (an organophosphate) was studied. For the evaluation of cardiotonic activity, Syme’s technique is
being employed, digoxin and propranolol were used as standard drug and β-blocker respectively to characterize
the effects on the receptors. The isolated perfused frog heart and hypodynamic frog heart showed dose
dependent positive ionotrophic effects. The parotoid gland secretion exihibited cardiac stimulant activities. The
propranolol was not able to block the effects of paraoxon induction on toad secretion. Thus, the present
investigation reports that the parotoid gland secretion increased the force of contraction, heart beat and
cardiac output in perfused frog’s heart, whereas, there was no change on hypodynamic heart, indicating that
there may be existence of two components, one with β–receptor stimulating activity and other acting directly on
the heart (independent of β1-adrenoreceptors).
KEY WORDS: Bufo melanostictus, Rana tigrina, Cardiotonic activity, Parotoid gland secretion, Paraoxon.
I.
INTRODUCTION
In recent years there has been an increased interest in search of new pharmaceutical compounds of
natural origin has intensified and been extended to include sources other than plant material [1].The
alarm/defense secretions of Amphibians also exhibit a number of pharmacological activities and quite a good
number of pharmacologically active substances like pahutoxin [2], bufotenin and bufotoxins [3], biogenic
amines [4], bioactive peptides [5,6 and 7] have been isolated in purified form from the skins of amphibians.
Amphibian skin is characterized by the presence of cutaneous glands spread over the body. Basically
toads have two types of alveolar glands in the epidermal layer of their skin i.e. (i) mucous glands and (ii)
granular glands [8, 9]. Mucous glands secrete mucus, functioning as a lubricant in the water to keep skin moist
and necessary for cutaneous respiration and protect the skin from mechanical damages and prevent microbial
settlement on the skin. Skin plays an important role in defense mechanism and mucus known to be
glycoproteinaceous being rich in a variety of protein residues such as mucin, musinigen, and carbohydrate
residues such as galactose, fukosa, and sialaic acid. Granular glands are also called poison glands which secrete
serous that provide protection from predators such as birds, mammals, snakes and crocodiles [10].
The parotoid gland of the Bufo species has been known to secrete several bio active compounds like
bufotenins, bufalins and bufotoxins [3] .The venomous secretions of these glands were also used as cardiotonic
drugs by Chinese and Japanese physiscians in folk medicine. The toad venom especially bufotenin secreted by
these glands was subject for mythological, pharmacological and medicinal practices for centuries [11]. The
Scientific research has confirmed the presence of bufalin in “Chan Su” a traditional Chinese medicine prepared
from dried white secretion of the skin glands of Chinese toad used as a drug for treating heart disorders,
heamorrhage of gums, sinusitis and other systemic illnesses [12].
In view of the importance of compounds present in the skin secretions of parotoid glands to
pharmacology, especially to heart physiology, an investigation was carried out on the effects of the parotoid
gland secretion of toad Bufo melanostictus, on the isolated hearts of the frog Rana tigrina.
61
Cardiotonic activity of Parotoid gland…
II.
MATERIALS AND METHODS
2.1 Studies on Heart beat:
The toads (7cm to 10 cm in length, weighed about 50 to 70 gm.) were collected from the vicinity of
Kakatiya University hostel buildings, Warangal, A.P., India. The parotoid glands were gently pressed with the
help of sterile forceps to release the secretions according to Meyer and Linde (1971) [13] and these secretions
were collected into ice-jacketed containers. The secretions were weighed to nearest milligram. They were stored
in cool conditions until further analysis. The secretion and the glands were weighed to the nearest milligram and
were homogenized (10%) in cold 0.9% of normal saline or frog Ringer's solution. The secretions were vortexed
for 5 minutes and centrifuged at 2000 rpm for 20 minutes. The freshly collected compounds were dissolved in
normal saline/frog ringer’s solution to study the effect on isolated hearts of freshly killed Rana tigrina. The
isolated heart mounted on to Syme’s cannula [14] was used for the study.
2.2 Preparation of isolated hearts for experiment:
Frog (Rana tigrina) was stunned by head-blow using a steel rod and pithed. The skin and abdomen
were cut and opened. The pectoral girdle was cut using a bone cutter and pericardium was cut and removed.
Syme`s cannula was connected to the reservoir of frog Ringers solution and introduced immediately into the
Sinus venosus of the heart. Frog Ringer's solution was immediately introduced into the sinus venosus of the
heart through posterior venacava after pericardium and the connecting blood vessels were removed and the heart
was removed from the animal and mounted on to the stand. The heart was covered with thin layer of cotton and
was wetted continuously with frog Ringer's solution to prevent the drying of the tissue. The heart was connected
to the starting lever, which was in turn connected to kymograph drum for recording the heart beat. The flow of
Ringer's solution into Syme’s cannula was maintained by fixing a glass tube into the cork fixed to the reservoir
(Marriott bottle) tightly. The heart was allowed to stabilize and when the heart rate and cardiac output were
stabilized, the recordings made on a slowly rotating drum to which sooted kymograph paper was affixed. The
effects of parotid secretion per se (in Ringer) and its fraction were studied on isolated perfused frog hearts. The
parameters studied included the force of contraction, heart rate and cardiac output. The volume of frog Ringer's
solution coming out of the heart per minute (Cardiac output) was measured by collecting the solution into a
measuring jar. The heart rate was measured by counting the number of heart beats per minute. Minimum 5 min
time was allowed between the additions of parotoid exudates per se (in Ringer) and its fraction. When blocker
was used, it was diluted with known amount of the physiological solution in syringe itself and added slowly.
The heart rate (HR), cardiac output (CO) and force of contraction were the parameters used for the study. The
solutions of parotoid gland secretion per se and the extracted fractions were prepared in frog Ringer’s solutions.
No suspending agents were used. The heart was constantly moistened with frog’s Ringer solutions from time to
time.
2.3 Hypo Dynamic Frog’s Heart
An isolated frog heart preparation as described under Syme’s technique was set up. Instead of one
reservoir, two reservoirs each for ½ calcium and full calcium were used. The levels in the reservoir maintained
constantly, which was tested by connecting each of the reservoirs to the Syme’s cannula.
Experiments were conducted by rendering the frog heart hypodynamic by letting into heart, frog
ringer’s containing ½ calcium from another reservoir through syme’s cannula. Force of contraction was
monitored to give half the magnitude of normal force of contraction. The effects of parotoid gland secretion on
hypo dynamic hearts were determined by using frog Ringer with ½ calcium concentration. Propranolol, the non
specific β-blocker was used to characterize the effects on the receptors.
Studies were conducted on 6 animals and each time collections from individual toads were used to see
the reproducibility of the effects. Samples were given as spot dose into Syme's cannula through a tuberculin
syringe. The heart rate and the cardiac output were measured simultaneously. Dose-dependent effects of the
parotoid gland secretion were noted to get optimum results.
2.4 Preparation of digoxin solution:
The marketed digoxin gift samples (Sunpharma Ltd.) were obtained from local market. Various
different dilutions were made with distilled water and labeled as follows, B1‐ 25 μg/ml, B2‐ 50 μg/ml. above
prepared samples were evaluated for their cardiotonic activity and treated as standard.
62
Cardiotonic activity of Parotoid gland…
2.4 Composition and Preparation of hypodynamic Ringer’s solution
Hypodynamic ringer solution was prepared by using standard method [15].
Sr. No.
Ingredients
Quantity
1
Sodium chloride (NaCl)
6.5 gm
2
Potassium chloride (KCl)
0.14 gm
3
Calcium Chloride (CaCl 2 )
0.03 gm
4
Sodium bicarbonate (NaHCO 3 )
0.2 gm
5
Glucose
2.0 gm
6
Distilled Water
1000 ml
2.5 Preparation of OP Compound concentrations for induction to study Pharmacological studies [16].
To observe the pharmacological effects after exposure of OP compound, Paraoxon (0, 0-di-ethyl-4nitrophynyl phosphate (2X10-5M)), concentration in the parotoid gland secretion and its extract. The OP
compound concentrations and normal saline were induced sub-cutaneously into parotoid gland contra laterally.
The in vivo effects of Paraoxon, on cardiotonic activity of parotoid gland secretion and its extract were noticed
after 4 hours time interval.
III.
RESULTS AND DISCUSSION
The results on cardiotonic activity of paraoxon induced and normal parotoid gland secretions (without
induction) are presented in Tables 1&2, Fig 1&2 and Graphs.1&2. The results indicated that the parotoid
exudates on isolated perfused frog hearts elicited dose dependent effect. There was an increase in force of
contraction (the positive ionotropic effect) per se (in ringer) and slight increase in cardiac output (Tables, Fig
and Graphs.1&2). Propranolol was not able to block the response of parotoid exudates per se (in Ringer) totally
(Figure. 1&2). A partial reduction in amplitude of contraction was noticed (Fig-1&2), indicating that there may
be two components, one with β–receptor stimulating activity and other acting directly on the heart (independent
of β1-adrenoreceptors). The gland extracts exihibit the cardiac stimulant activities (Fig-1&2). The dose
dependent changes were measured in heart rate (HR), cardiac output (CO) and percentage of increase in force of
contraction (% IFC). In all these dose concentrations Parotoid gland exudates exhibited prominent cardio tonic
effect in hypodynamic heart. Propranalol was not able to block the total amplitude of force of contraction but
reduction in the force of contraction indicates dual roles like cardio tonic and cardiac stimulant activities.
On hypodynamic heart normal parotoid gland secretion showed dose dependent changes in heart rate
(HR), cardiac output (CO) and percentage of increase in force of contraction (% IFC). The normal parotoid
gland secretion showed dose dependent changes on isolated heart, and the results were observed as 42
beats/min (HR), 10 ml/min (CO). At 0.1 ml (50µg) dose, the heart rate was noted as 40 beats/min (HR), cardiac
output of 12 ml/min (CO) and with an increase of 50% in force of contraction (% IFC). At 0.2 ml (100µg) dose,
the results obtained were 38 beats/min (HR), 15 ml/min (CO) and 70% of increase in force of contraction (%
IFC). At 0.3 ml (150µg) dose, results were 36 beats/min (HR), 16 ml/min (CO) and 90% of increase in force of
contraction (% IFC), while at the standard digoxin dose (1000ng), the results were 38 beats/min (HR), 16
ml/min (CO) and 30% increase in force of contraction (% IFC). At the maximum concentration of propranolol
and the maximum dose of parotoid gland secretion i.e., 0.3 ml (150µg), the results obtained were 38 beats/min
(HR), 14 ml/min (CO) and 20% increase in force of contraction (% IFC) which revealed that the Propranolol
was not able to block the response of parotoid exudates per se (in Ringer) totally.
The bufotenins, bufalins, bufotoxins of toads [3] and bufalins present as free genins or as conjugates of
steroids in these secretions were reported to be similar to digitalis in their action. Further studies [17], suggested
that the cardiotonic compounds affect the force of contraction without affecting the heart rate significantly. The
results obtained in the present investigation are consistent with above observations. The parotoid gland extracts
exhibit the cardiac stimulant activities [18, 19] and the OP compounds like Paraxon could not inhibit the
cardiotonic activity of parotoid gland exudates. OP compounds inhibit the level of biochemical constituents and
esterase activities [20].
3.1 The effect of Paraoxon
The effect of paraoxon (OP) induced parotoid gland secretion revealed that the cardiac output and
increase in force of contraction (the positive inotropic effect) per se (in ringer) increased on isolated heart and
hypodynamic heart (Table, Fig and Graph-2) compared to normal parotoid gland secretion (Table, Fig and
Graph-1). The effect of Paraoxon induced parotoid gland could not decrease cardio tonic and cardiac stimulant
activities of parotoid gland secretion. Propranolol was unable to block the amplitude of force of contraction in
the Paraoxon induced parotoid gland secretion (Fig-2) but increase in activity compared to normal parotoid
gland secretion (Figure-1) was noticed.
63
Cardiotonic activity of Parotoid gland…
On hypodynamic heart paraoxon induced parotoid gland secretion showed dose dependent changes in
normal and the results were observed as 55 beats/min (HR), 12 ml/min (CO), while paraoxon induced parotoid
gland secretion showed dose dependent changes at 0.1 ml (50µg) with 53 beats/min (HR), 14 ml/min (CO) and
50% increase in force of contraction (% IFC) while at 0.2 ml (100µg) dose results were 50 beats/min (HR), 16
ml/min (CO) and 70% increase in force of contraction (% IFC). At the 0.3 ml (150µg) dose, the results were 50
beats/min (HR), 18 ml/min (CO) and 90% increase in force of contraction (% IFC). At the standard digoxin
dose (1000ng) results were observed to be 48 beats/min (HR), 20 ml/min (CO) and 20% increase in force of
contraction (% IFC). The Propranolol and paraoxon (the maximum dose) induced parotoid gland secretion, i.e.,
at 0.3 ml (150µg) results obtained were 54 beats/min (HR), 14 ml/min (CO) and 40% increase in force of
contraction (% IFC) reveals that the Propranolol was not able to block the response of parotoid exudates per se
(in Ringer) totally (Fig, Table & Graph-1&2).
Toad-toxins act by inducing various physiological effects on higher as well as lower vertebrates.
Granular gland secretions when entered into stomachs of higher-vertebrates cause nausea, weakening of
respiration and muscular paralysis, while in contact with eyes they produce serious inflammations [21]. Toad
secretions may also boast of adrenalin, which is a result of chemical change within mature secretion [22]. Skin
Clinical aspects of toad-toxins have been studied particularly in Bufo marinus. Secretions of B. marinus are
cardio active, due to activity of bufogen and bufotalin exhibiting clinical symptoms like dermatitis, hypotension
and severe arrhythmia [23]. Toad-secretions have also been found to show second-degree Wenckebach atrio
ventricular block and T-wave change [24].
Bufogenines are the inhibitors of the enzyme Na + /K + ATPase activity in the myocardial cell memebrane
[25] acting evidently through the same mechanism. Bufonid toad’s skin of the genera Atelopus,
Dendrophryniscus and Melanophryniscus contain Bufadienolides or related compounds on basis of assays of
inhibition of Na + /K + ATPase or binding of titrated ouabin [25].
IV.
CONCLUSION
It will be interesting to isolate the active chemical constituents which are responsible for the
cardiotonic activity as well as to determine the possible mechanism of action. It is possible that the peptide or an
amine or may be the other compound effecting the cardiac activity.
Table-1 Effect of parotoid gland secretion on isolated frog heart (the parameters include the force of
contraction, heart rate and cardiac output.)
Dose
On Normal Heart
Normal
0.1 ml(50 µg)
0.2 ml(100 µg)
0.3 ml(150 µg)
Digoxin 400 ng
Digoxin 1000 ng
On Hypodynamic heart
Normal
0.1 ml(50 µg)
0.2 ml(100 µg)
0.3 ml(150 µg)
Digoxin 1000 ng
PP+0.3ml(150 µg)
Normal
HR (beats/min)
CO (ml/min)
48
50
52
54
48
48
12.5
13
13
13
12
14
42
40
38
36
38
38
42
10
12
15
16
16
14
11
64
Result
Increase in force of
contraction (in mm)
5
3
2
0.5
1
5
7
9
3
2
-
% Increase in force
of contraction
50
30
20
5
10
50
70
90
30
20
-
Cardiotonic activity of Parotoid gland…
Fig-1 Effect of parotoid gland secretion on isolated frog heart the parameters includes the force of
contraction, heart rate and cardiac output.
Graph-1 Effect of parotoid gland secretion on isolated frog heart elicited a dose dependent increase in
force of contraction (positive inotropic effect).
Table-2 Effect of Paraoxon induced parotoid gland secretion on isolated frog heart the parameters
includes the force of contraction, heart rate and cardiac output
Dose
Result
On Normal Heart
HR (beats/min)
CO (ml/min)
Normal
0.1 ml(50 µg)
0.2 ml(100 µg)
0.3 ml(150 µg)
Digoxin 400 ng
Digoxin 1000 ng
60
58
56
58
60
56
Normal
0.1 ml(50 µg)
0.2 ml(100 µg)
0.3 ml(150 µg)
Digoxin 1000 ng
PP+0.3ml(150 µg)
Normal
55
53
50
50
48
54
58
12
14
16
16
12
14
On Hypodynamic heart
11
14
16
18
20
14
12
65
Increase in force of
contraction (in mm)
% Increase in force
of contraction
2
4
5
1
1
20
40
50
10
10
5
7
9
2
4
-
50
70
90
20
40
-
Cardiotonic activity of Parotoid gland…
Fig-2 Effect of Paraoxon induced parotoid gland secretion on isolated frog heart the parameters includes
the force of contraction, heart rate and cardiac output.
Graph-2.Effect of Paraxon induced parotoid gland secretion on isolated frog heart elicited a dose
dependent increase in force of contraction (positive inotropic effect).
V.
ACKNOWLEDGEMENTS
The authors are thankful to the authority of University grants commission, New Delhi for financial
assistance under major research project F.No.39-596/2010(SR) and also to the Head, Department of Zoology
and Principal, University college of Pharmaceutical Sciences, Kakatiya University, Warangal, for providing
laboratory facilities.
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